Hi,I have an insert cloned into TOPO vector and I want to interrupt this insert with an antibiotic resistance gene.I cut the insert cloned in TOPO vector with NruI and NheI. The restriction resulted in 5kbp fragment and a small 700bp fragment. I also cut the vector that has the resistance marker gene with EcoRV and XbaI. The restriction resulted in two closed fragments around 3kbp, the fragment I need is the largest.I purified the TOPO/insert vector and the 3kbp fragment and I set up a ligation reaction. The insert (around 3kbp) : vector (around 5kbp) molar ratio was 5:1, and the ligation reaction was 16 hours 15ºC.This reaction is a blunt (NruI-EcoRV) – cohesive (NheI-XbaI, compatible ends) one.When I saw my ligation product in agarose gel, I observed new bands that seem to be that the ligation reaction worked. However when I transformed it in TOP10 F’ competent cells, I had no recombinants.The results have been always the same no matter what I change.I set up a control reaction, digesting the resistance gene/vector and re-ligating it, and in this case I did obtained recombinants, only if I did not purified the fragments digested. When I purified them, I didn’t obtained recombinants. So, it seems to be a purification problem, but I also think that is a ligation problem, and observing the bands in the agarose gel after ligation, I think that only one end is ligating. I have changed the band purification system using resin kits and electroelution. Do you think that a blunt-cohesive ligation reaction is too difficult to perform? What ligation conditions do you think I should use in this case (I’m using Invitrogen ligase, and I’m following the blunt conditions they recommend).

Thanks a lot for any suggestion

MM

-MM20-

Hi MM20,

Sorry I got a little lost in your explanation. Your 5 to 1 molar ratio was this calculated after gel purification or from the initial starting amount digested? I firmly believe that gel purification methods are not efficient unless there is ample DNA to start with. What I would want to do (and you may have already done this) is visualise the DNA on a gel after purification and before ligation from this visualisation I would then set up a number of ratios ranging from 1 to 1, 5 to 1 and 10 to 1 (insert to vector). I would also try to keep the concentrations on the higher end of the spectrum.

Hope this helps,

Scott

-Scott-

Hi Scott,The molar ratio was calculated after gel purification, just by visualising the DNA on a gel. I’ll try with other ratios.Thanks a lotMM